α cyano 4 hydroxycinnamic acid hcca matrix solution

Sample preparation was done following a protocol described by Shevchenko et al. [20 (link)] with minor modifications. IgE reactive spots corresponding to 2D blot were excised with sterile scalpels and destained in 50 mM ammonium bicarbonate (ABC) (pH 8.0) containing 50 % ethanol followed by reduction and alkylation with 10 mM DTT (Sigma) and 55 mM iodoacetamide (Sigma) respectively. After that gel pieces were rehydrated and dehydrated in 50 mM ABC and 100 % acetonitrile (ACN) respectively. In-gel digestion was carried out in 12.5 ng/μl of trypsin (trypsin Gold, Promega, USA) at 37 °C for 16 h. After removing trypsin, peptides were extracted from gel pieces by vigorous vortexing in extraction buffer composed of 30 % ACN and 1 % Trifluoroacetic acid. The extracted peptides were dried in a speed vac (Savant, USA). About 1.5 μl peptide digests were mixed with equal volume of 0.5 mg/ml α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (Bruker Daltonics, Germany) and was dotted onto MTP 384 ground steel target plate (Bruker) and air dried.

Fresh bacterial colony growth was deposited on a polished steel MSP 96 target (Bruker Daltonics, Leipzig, Germany) and covered with 1 μL of a 70% formic acid solution. Following air drying, the bacterial spot was overlaid with 1 μL of a saturated α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (Bruker Daltonics). Mass spectra were acquired and analyzed using a microflex LT mass spectrometer (Bruker Daltonics) in combination with research-use-only (RUO) versions of the MALDI Biotyper software (MBT Compass v4.1) and the reference database v9.0.0.0 (8,468 spectra covering 2,969 species). Calibration was done by following the manufacturer's instructions and using the manufacturer's recommended bacterial test standard. Bacterial species were assigned for scores of ≥2.0.

The bacterial cell pellet of the second aliquot was dissolved in 50 μL of formic acid 70% by vortexing; then, 50 μL of pure acetonitrile was incorporated into the solution, vortexed, and centrifuged (10,000 g for 2 min) for the following direct MALDI-TOF MS identification assay. In brief, 1 μL supernatant was spotted onto a target plate, and 1 μL of an α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (Bruker Daltonik GmbH, Bremen, Germany) was added and air dried at room temperature. Identifications were performed by the Bruker microflex MALDI-TOF MS system using the MALDI Biotyper 3.0 RTC database (Bruker Daltonik GmbH, Bremen, Germany). According to the manufacturer, a score of ≥2.0 indicates reliable identification at the species level, while a score between 1.7 and 1.99 indicates a reliable identification at the genus level, and a score below 1.7 indicates a NRI. Thus, these were the criteria used when comparing the results of the direct MALDI-TOF MS identification assay with those from the conventional culture-dependent identification for the evaluation of direct identification performance.